Vaccinal preparations

ABSTRACT

Vaccinal preparations comprising an antigen such as tumor-associated antigens and viral antigens, together with a hydrophobized polysaccharide as adjuvant, e.g., hydrophobized polysaccharides containing a saccharide unit whose primary hydroxyl group is represented by the following formula: —O—(CH 2 ) m CONH(CH 2 ) n NH—CO—O—R wherein R represents an alkyl group or a sterol residue; m represents 0 or 1; and n represents an arbitrary positive integer, at a ratio of one to five units per 100 saccharide units that constitute the polysaccharide, preferably comprising an antigen encapsulated in microparticles of aggregated hydrophobized polysaccharide.

TECHNICAL FIELD

The present invention relates to a vaccinal preparation comprising anantigen and a hydrophobized polysaccharide. More specifically, itrelates to a vaccinal preparation comprising antigens such astumor-associated antigens and virus antigens, together with ahydrophobized polysaccharide which acts as an adjuvant, wherein theantigens and the hydrophobized polysaccharide form a complex. Thevaccinal preparation of the present invention has, in particular,activating and inducing effects on cytotoxic T cells (abbreviated as“CTL” hereinafter in the specification) that are specific for theantigen, and therefore, the preparation is extremely useful as a vaccinefor preventive and/or therapeutic treatment of cancers, viral diseasesand autoimmune diseases.

BACKGROUND ART

CTLs have actions of specifically recognizing cancer cells orvirus-infected cells through their T cell receptors or other, anddestroying or damaging the cells. Their roles are important as abio-defense mechanism against cancers or viruses. Recently, it has beenrevealed that T cell receptors of CTL do not directly recognize thespecific antigen expressed on the surface of cancer cells orvirus-infected cells. Instead, they recognize complexes including an MHCclass I antigen, which is expressed on the surface of antigen-presentingcells, per se, such as macrophages, dendritic cells, cancer cells orvirus-infected cells, together with an oligopeptide derived from thespecific antigen bound to the MHC class I antigen (“CTL epitope” of thespecific antigen).

It is considered that the complex of an MHC class I antigen/anoligopeptide derived from a specific antigen is formed by a processingas explained below. Antigenic proteins are synthesized in cytoplasm, andthen some of the proteins are degraded into oligopeptides byintracellular protease complexes (“proteasome”). Some of theoligopeptides (consisting of 9-10 amino acid residues) are thentransported from cytoplasm into the endoplasmic reticulum membrane bytransporter proteins (TAP: transporter in antigen processing) that arepresent in the endoplasmic reticulum membrane, and oligopeptides havinghigh affinity for the MHC class I antigen are preferentially bind to theMHC class I antigen and presented on the surfaces of the cells.

It has been attempted to develop vaccines that artificially can activatethe CTL (CTL-activating vaccines) in order to provide a method ofpreventive and/or therapeutic treatment of cancers, viral diseases orautoimmune diseases by eliminating cancer cells, virus-infected cells orautoantigen-reactive lymphocytes. To achieve the aforementioned purpose,it is necessary to perform immunization of a living body with cancercells or virus-infected cells, per se, which expresses specificantigens, or alternatively, to introduce the specific antigens or theoligopeotides derived therefrom into the processing machinery for theantigen presenting cells so that a complex with the MHC class I antigencan be expressed.

In order to develop such “CTL-activating vaccines”, some attempts weremade which include, for example, 1) a method comprising the step oftransforming a gene encoding a specific antigenic protein by means of aviral vector; 2) a method comprising the step of introducing a specificantigenic protein having an appropriate molecular weight and several CTLepitopes into cytoplasm by means of a certain technique, and 3) a methodcomprising the step of binding an oligopeptide consisting of 9-10 aminoacid residues as a potential CTL epitope directly to an MHC class Iantigen of an antigen presenting cell.

Among them, method 1) is a so-called gene therapy, whose efficacy andsafety have not yet been established. Efficacy obtained by using method3) have been shown in animal studies; however, practical problems mayarise for applications to humans. Since almost every patient hasdifferent kind of MHC class I antigens, it should be necessary to coverthe diversity of CTL epitopes corresponding to the variety of antigens.In other words, it is necessary to elucidate amino acid sequentialmotifs of oligopeptides having high affinity for each kind of the MHCclass I antigens, and to provide customized oligopeptides as medicamentsthat correspond to each of the MHC class I antigen. Therefore, thedevelopment of vaccines based upon this class will be extremelydifficult.

As to the approach according to method 2), some successful examples areknown and its efficacy and safety seem satisfactory. Therefore, themethod has been expected as a most successful means to develop the CTLactivating vaccines applicable to variety of patients. However, when aspecific antigenic protein, per se as a polypeptide, is administered toa living body to activate specific CTLs, the protein is usuallyadministered as a mixture with an adjuvant. For example, ISCOM(Takahashi et al., Nature, 344, 873-875, 1990), QS-21 (Newman et al., J.Immunol., 148, 2357-2362, 1992), mannan-coated liposomes (WO92/4887), AF(Raychaudhuri et al., Proc. Natl. Acad. Sci. USA, 89, 8308-8312, 1992)or other have been reported as such adjuvant.

It is known that polysaccharide/cholesterol derivatives can be used aspolysaccharide coatings for liposomes [Japanese Patent UnexaminedPublication (KOKAI) No.(Sho)61-69801/1986], or coatings for lipidemulsions [Japanese Patent Unexamined Publication (KOKAI)No.(Sho)63-319046/1988]. As explained above, it is also known thattumor-associated antigens or virus antigen/liposome complexes, coatedwith polysaccharides containing mannose, have inducing activity of CTL(WO92/4887).

However, all of the aforementioned products should be manufactured by acoating process using a polysaccharide/cholesterol derivative after aliposome formation or a lipid emulsification of an antigenic protein (anantigen). As for a complex consisting solely of apolysaccharide/cholesterol derivative and an antigenic protein (anantigen), anything has not been reported as to what effects can bespecifically obtained by the complex.

As mentioned above, in order to activate specific CTLs by administeringa kind of antigenic protein as a polypeptide, to human, it is necessaryto use a certain adjuvant. However, no adjuvant has been available whichachieves efficient immunization and safety, and can be readily prepared.

DESCRIPTION OF THE INVENTION

The inventors of the present invention conducted various studies todiscover a substance which can be used as a simpler and more efficientadjuvant. As a result, they found that hydrophobized polysaccharidescomposed of naturally occurring polysaccharides modified with alkylgroups or cholesterol groups can encapsulate, quite readily andefficiently, tumor-associated antigenic proteins or virus antigenicproteins to form complexes without any need of phospholipids. They alsofound that specific CTLs were activated in animals administered with thecomplex and bio-defense reactions were induced. The present inventionwas achieved on the basis of these findings.

The present invention thus provides vaccinal preparations comprising ahydrophobized polysaccharide and an antigen, and vaccinal preparationscomprising a complex of a hydrophobized polysaccharide and an antigen.According to preferred embodiments of the present invention, there areprovided the aforementioned vaccinal preparations which contain anantigen encapsulated in microparticles comprising aggregatedhydrophobized polysaccharides; and the aforementioned vaccinalpreparation comprising the hydrophobized polysaccharide as an adjuvant.Those vaccinal preparations can be used for, for example, anti-cancerand anti-viral or autoimmune disease therapies.

According to preferred embodiments of the present invention, there areprovided the aforementioned vaccinal preparations wherein thehydrophobized polysaccharide is a polysaccharide modified with an alkylgroup or a sterol residue; the aforementioned vaccinal preparationswherein the hydrophobized polysaccharide is characterized to contain asaccharide unit, at a ratio of one to five per 100 saccharide units thatconstitute the polysaccharide, whose primary hydroxyl group is a grouprepresented by the following formula (I):

—O—(CH₂)_(m)CONH(CH₂)_(n)NH—CO—O—R  (I)

wherein R represents an alkyl group or a sterol residue; m represents 0or 1; and n represents an arbitrary positive integer; the aforementionedvaccinal preparations wherein the polysaccharide is pullulan or mannan;and the aforementioned vaccinal preparations wherein the sterol residueis cholesterol residue.

According to further preferred embodiments of the present invention,there are provided the aforementioned vaccinal preparations wherein theantigen is a protein which is presented as an oligopeptide by an MHCclass I antigen and induces cytotoxic T-cells; the aforementionedvaccinal preparations wherein the antigen is ErbB-2 protein; and theaforementioned vaccinal preparations wherein the antigen is atumor-associated antigen, a viral antigen, or an autoantigen-reactiveT-cell receptor.

As another aspect of the present invention, there are provided methodsfor immunization which comprises the step of administering to a mammalthe aforementioned vaccinal preparations; and the aforementioned methodswhich are used for inducing antigen-specific cytotoxic T-cells. As afurther aspect of the present invention, adjuvants comprising ahydrophobized polysaccharide are provided. As a preferred embodiment ofthe aforementioned invention, the adjuvants are provided which are usedfor the formulation of a vaccinal preparation together with an antigen.There is also provided a use of a hydrophobized polysaccharide for themanufacture of the aforementioned vaccinal preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the growth curves of tumor cells on the mice immunizedwith preparations including a complex comprising cholesterol-modifiedmannan or cholesterol-modified pullulan and recombinant protein derivedfrom human ErbB-2 oncogene then inoculated with human ErbB-2 expressing(CMS7HE) or non-expressing (CMS7neo) mouse cancer cells. The verticalaxis represents the volume of the tumors (mm³) calculated by approximatecalculation using measured longitudinal and latitudinal length of thetumor, and the horizontal axis represents days after the tumorinoculation. In the figure, No. 1 to 3 show the data for the micetreated with CHM-erbB2, CHM-CAB (control group) and CHP-erbB2,respectively.

FIG. 2 depicts the growth curves of tumor cells on the mice immunizedwith preparations including a complex comprising cholesterol-modifiedmannan or cholesterol-modified pullulan and recombinant protein derivedfrom human ErbB-2 oncogene then inoculated with human ErbB-2 expressing(CMS7HE) or non-expressing (CMS7neo) mouse cancer cells. The verticalaxis represents the volume of the tumors (mm³) calculated by approximatecalculation using measured longitudinal and latitudinal length of thetumor, and the horizontal axis represents days after the tumorinoculation. In the figure, No. 4 to 7 show the data for the micetreated with CHP-CAB (control group), ErbB-2 protein only (controlgroup), CAB only (control group) and non-immunized (naive) mice,respectively.

FIG. 3 shows the cytotoxic activity of the spleen cells collected frommice immunized under the same conditions as those in FIG. 1. The spleencells were cultured in vitro with stimulated with human ErbB-2expressing Balb/c mouse derived CMS17HE cells, and their cytotoxicityagainst ⁵¹Cr-labeled target cells was measured (the sequential numbers 1to 7 in FIG. 3 corresponds to the numbers of the immunization groupdefined in FIGS. 1 and 2). In the figure, the vertical axis representscytotoxicity calculated by subtracting the fraction of the non-specificleak of ⁵¹Cr from the labeled cells, and the horizontal axis representsthe ratio of the number of the spleen cells to that of the target cells.

FIG. 4 depicts the differences of human ErbB-2 specific CTL activitieswhen monoclonal antibodies recognizing various mouse immunocyte-surfacemarkers were added at a concentration of 1:2 dilution ratio under thesame conditions as the CHM-erbB2 immunized group in FIG. 3. In thefigure, No. 1 represents the data obtained in the absence of anyantibodies, No. 2 in the presence of anti-CD3 antibody, No. 3 in thepresence of anti-CD4 antibody, No. 4 in the presence of anti-CD8antibody, No. 5 in the presence of anti-Kd antibody, No. 6 in thepresence of anti-Dd antibody, No. 7 in the presence of anti-Ld antibody,and No. 8 in the presence of anti-MHC class II antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

As used herein, the term “adjuvant” means a substance which is usedtogether with an antigen to modify an immune response against theantigen, and the term includes those used generally for enhancingantibody production and cellular immunity, as well as those optionallyused for changing immune response in its nature.

<1> Antigen

The antigens used according to the present invention are notparticularly limited so long as they induce immunity. For example,polypeptides, polypeptide complexes, glycoproteins, nucleic acids andother may be used. The antigen may be a pathological target, per se,such as viruses or cancer cells, or a part thereof. Alternatively, theantigens may be those produced by pathological tissues such asneoplastic tissues and virus-infected cells. According to the presentinvention, antigenic proteins, in particular, those presented asoligopeptides by MHC class I antigens and inducing CTLs may preferablybe used. The antigenic proteins are not limited so far that they containone or more antigenic determinants, and their origin and purity are notparticularly limited. However, for example, those produced by geneticrecombinant techniques are preferred. Molecular weight of the antigenicproteins may generally be from about 500 to 100,000, preferably fromabout 2,000 to 100,000. More specifically, examples include oncogeneproducts, ErbB-2 protein, ras p21 protein, tumor suppressor gene productp53 protein, virus derived protein, T cell receptors such asautoantigen-reactive (i.e., autoimmune disease-inducing) T cellreceptors, and proteins containing a partial sequence thereof.

<2> Hydrophobized Polysaccharide

The hydrophobized polysaccharides or the microparticles comprisinghydrophobized polysaccharide aggregates can be produced by knownmethods, for example, those described in Akiyoshi et al.,Macromolecules, 6, pp.3062-3068, 1993; Akiyoshi et al., J. Proc. Japan.Acad., 71, 71B, p.15, 1995; Japanese Patent Unexamined Publication(KOKAI) Nos.(Sho)61-69801/1986, (Hei)3-292301/1991, (Hei)7-97333/1995and other.

Polysaccharides constituting the hydrophobized polysaccharide are notparticularly limited so long as they are polymers composed of saccharideresidues linked by glycoside bonds. Examples of the saccharide residuesconstituting the polysaccharides include, for example, monosaccharidessuch as glucose, mannose, galactose, and fucose, and those derived fromdisaccharides, oligosaccharides and the like. The saccharide residuesmay be linked with 1,2-, 1,3-, 1,4- or 1,6-glycoside bond, and the bondmay be either in α- or β-linkage. The polysaccharide may be linear orbranched. Glucose residue is preferred as the saccharide residue. As thepolysaccharide, naturally occurring or synthesized polysaccharides suchas pullulan, dextran, amylose, amylopectin, mannan and the like,preferably mannan and pullulan may be used.

As the hydrophobic group, for example, mono-chain or bi-chain alkylgroups or sterol groups are preferably introduced at a ratio of 1 to 5modifications per 100 monosaccharide units (5% by weight or less).However, the hydrophobic groups are not limited to those mentionedabove, and those achieving high encapsulating ratio can be appropriatelychosen depending on the molecular weight, isoelectric point and other ofan antigen to be encapsulated. As the sterol residue, for example,cholesterol, stigmasterol, β-sitosterol, lanosterol, ergosterol residuesand others can be used, and cholesterol residue can be preferably usedas the sterol residue. As the alkyl group, those having 20 or less ofcarbon atoms, more preferably 10-18 carbon atoms can be used, and theymay be linear or branched.

As the hydrophobized polysaccharide, a preferred example includes thosecontaining a saccharide unit, at a ratio of one to five per 100saccharide units that constitute the polysaccharide, whose primaryhydroxyl group is represented by the following formula (I):

—O—(CH₂)_(m)CONH(CH₂)_(n)NH—CO—O—R  (I)

wherein R represents an alkyl group or a sterol residue; m represents 0or 1; and n represents an arbitrary positive integer. As the alkyl groupand the sterol residue, those mentioned above may preferably be used.The symbol “n” may preferably be from 1 to 8. As the hydrophobizedpolysaccharide, those formed by means of linkers such as those describedin Japanese Patent Unexamined Publication (KOKAI) No.(Hei)7-97333/1996may also be used.

<3> Method for Encapsulating Antigens by Using the HydrophobizedPolysaccharide

The complexes comprising the hydrophobized polysaccharide and theantigen can be isolated and purified by mixing microparticles ofaggregated hydrophobized polysaccharide and antigens at roomtemperature, and then treating the mixture by gel chromatography(Nishikawa, Macromolecules, 27, pp.7654-7659, 1994). The resultingcomplexes of the hydrophobized polysaccharide and the antigen, per se,can be used as the vaccinal preparation of the present invention;however, they may optionally be subjected to further processes such assterilization by standard methods.

<4> Immunization with the Complex Comprising the HydrophobizedPolysaccharide and the Antigen

The vaccinal preparation of the present invention may be administered toan animal at a given amount for immunization in the same manner as otherknown vaccinal preparations, and the titer of the vaccinal preparationcan also be determined similarly. The vaccinal preparation of thepresent invention is generally administered by subcutaneous injection.However, the routes of administration are not limited to thesubcutaneous injection. For example, other parenteral administrationssuch as intravenous and intramuscular administrations, and oraladministration can also be employed. Doses of the vaccinal preparationof the present invention to achieve immunization can be appropriatelychosen. For example, a general dose may range from about 50 to 150 μg asthe amount of an antigen per administration, and once to fouradministrations are preferred.

For the manufacture of the vaccinal preparation of the present inventionas a pharmaceutical preparation, conventional excipients, carriers anddiluents can be suitably used depending on the prescribed route ofadministration.

EXAMPLES

The present invention will be explained more specifically by referringto the following examples. However, the scope of the present inventionis not limited to the following examples.

Example 1 Preparation of a Complex of Cholesterol-modified Mannan orPullulan and Oncogene Product ErbB-2 Protein

Hydrophobized polysaccharides were synthesized according to the methodof Akiyoshi et al. (Akiyoshi et al., Macromolecules, 26, 3062-3068(1993)). As a cholesterol-modified mannan, mannan having a molecularweight of about 55,000 (Sigma) modified with about 2.3 cholesterolresidues per 100 monosaccharide units was obtained (abbreviated as“CHM-55-2.3” hereafter). As a cholesterol-modified pullulan, pullulanhaving a molecular weight of about 108,000 (Hayashibara BiochemistryInstitute) modified with about 0.9 cholesterol residues per 100monosaccharide was obtained (abbreviated as “CHP-108-0.9” hereafter). Inaqueous solutions, each aggregate of the hydrophobized polysaccharidescontained about 7 hydrophobic domains formed by 7 associated cholesterolresidues in average.

The above-obtained hydrophobized polysaccharides were dissolved in DMSO,dialyzed against PBS (pH 7.9), and then sonicated by using a probe typesonicator (URP, Tomy Seiko) at 40W for 10 minutes. These solutions werefiltered successively through membranes having a pore size of 1.2 μm,0.45 μm, and 0.2 μm to obtain aqueous solutions of microparticlescomprising aggregated hydrophobized polysaccharides. The concentrationof the hydrophobized polysaccharide of each solution was determined as9.62 mg/ml for CHP-108-0.9, and 6.97 mg/ml for CHM-55-2.3 by thephenol-sulfuric acid method.

A recombinant protein composed of a polypeptide corresponding to aminoacids 1 to 147 of the oncogene product human ErbB-2 protein (Coussens etal., Science, 230, 1132, 1985), whose N-terminus was fused to ahistidine hexamer, was expressed in Escherichia coli and used as antigenprotein. A cDNA comprising a region encoding a polypeptide correspondingto the amino acids 1 to 147 of the human ErbB-2 protein was amplified byPCR using the following two oligonucleotide primers:

HN40: 5′-AGCTGCAGTGATCACCATGGAGCT-3′(SEQ ID NO:1 in SEQUENCELISTING),and

HN51: 5′-TGAATTCTATGTGAGACTTCGAAGCTGCA-3′(SEQ ID NO:2 in SEQUENCELISTING)

The resulting 463 bp DNA fragment was blunt-ended by T4 DNA polymerasetreatment, and digested with a restriction endonuclease BclI. A plasmidexpression vector, pQE11 (Qiagen), was digested with a restrictionendonuclease HindIII, blunt-ended by using T4 DNA polymerase, andfurther digested with a restriction endonuclease BamHI. The resultingBamHI-ΔHindIII fragment of about 3.3 kbp derived from the pQE11 vectorwas ligated to the aforementioned PCR-amplified DNA fragment of thehuman ErbB-2 cDNA by using a ligation kit (Takara Shuzo), and used forthe transformation of E. coli strain JM109. Restriction maps wereprepared for ampicillin-resistant clones to chose correctly constructedclones. Then, according to the attached manual of Qiagen, a recombinantprotein was purified. From the recombinant microorganisms cultured in 1liter of a culture medium, about 20 mg of the recombinant protein wasobtained.

A solution of the human ErbB-2 recombinant protein (containing 2.0 mg/mlof the protein in PBS/6M urea) was added to the aforementioned aqueoussolution of hydrophobized polysaccharides and mixed to obtain acolorless and transparent solution containing the complex of thehydrophobized polysaccharides and the antigen (5 mg/ml of CHM or CHP,0.25 mg/ml of the human ErbB-2 recombinant protein). As a result of DLS(Dynamic Light Scattering) measurement of the solution, it was foundthat complex microparticles having the particle size of about 250 nm andk2/k12 ratio of 0.156 was obtained (the resulting complex of thecholesterol-modified mannan and the ErbB-2 protein will be abbreviatedas “CHM-erbB2”, and the complex of the cholesterol-modified pullulan andthe ErbB-2 protein as “CHP-erbB2”).

On the other hand, under a condition where the same buffer was used butin the absence of the hydrophobized polysaccharides, all the humanErbB-2 recombinant protein was insolubilized and deposited asprecipitates.

As a negative control for immunization experiments, a complex of eachhydrophobized polysaccharide and a protein was prepared by usingCarbonic Anhydrase II (Sigma, referred to as “CAB” hereinafter) (thecomplex of the cholesterol-modified mannan and CAB will be abbreviatedas “CHM-CAB”, and the complex of the cholesterol-modified pullulan andCAB as “CHP-CAB”).

Example 2 Vaccinal Action of CHM-erbB2 and CHP-erbB2 (CTL-inducingEffect and Anticancer Effect)

A suspension of CHM-erbB2 or CHP-erbB2 prepared in Example 1 wassubcutaneously administered twice with an interval of one week to femaleBalb/c mice, each group consisting of three mice, at the amountequivalent to 25 μg of the protein per mouse. Then, recombinant cellsCMS7HE, which were prepared by expressing neo gene as a selection markerand human ErbB-2 in fibrosarcoma cell strain deriving from a micesyngeneic with Balb/c mice (see, DeLeo et al., J. Exp. Med., 146, 720,1977) in a conventional manner, or recombinant cells CMS7neo, which wereprepared by expressing only the neo gene derived from the micefibrosarcoma cell line in a similar manner as a negative control, weresubcutaneously inoculated to the mice.

The size of the inoculated tumors was measured periodically. As shown inFIG. 1, the growth inhibition and complete regression of the tumorexpressing human ErbB-2 was observed in all of the three mice immunizedwith CHM-erbB2 or CHP-erbB2 (FIG. 1, Nos. 1 and 3, left columns). On theother hand, as to the inoculated tumors not expressing the human ErbB-2,no inhibitory effect on the tumor growth was observed in the mice evenwhen immunized with CHM-erbB2 or CHP-erbB2 (FIG. 1, Nos. 1 and 3, rightcolumns), which suggested antigen-specificity of the immunization. As tothe immunization with the recombinant human ErbB-2 (FIG. 2, No. 5) orwith unrelated CAB (FIG. 1, No. 2 and FIG. 2, Nos. 4 and 6), elicited notumor growth inhibitory effect.

Spleen cells were collected from the mice immunized in the same manneras described above, and stimulated with CMS17HE cells which wassyngeneic with Balb/c mice and expressing human ErbB-2, which had beentreated by radiation in vitro, and their cytotoxicity against⁵¹Cr-labeled CMS7HE cells, CMS17HE cells, CMS7neo cells, CMS17neo cellsand CMS7 cells was measured in a standard method. As shown in FIG. 3,only when the spleen cells obtained from the mice immunized withCHM-erbB2 or CHP-erbB2 were used, the human ErbB-2 expressing cells(CMS7HE; CMS17HE cells were used as a positive control because the cellswere used for the stimulation) were remarkably damaged or killeddepending on the ratio of the target cell number and the spleen cellnumber, compared to the non-expressing cells (CMS7neo, CMS17neo andCMS7) (FIG. 3, Nos. 1 and 3).

In the above experiments, when monoclonal antibodies for various mouselymphocyte surface markers were added at a concentration of 1:2 dilutionduring the course of the assay for the human ErbB-2 specific CTLactivity of the spleen cells obtained from CHM-erbB2 immunized mice,only the antibodies against Kd inhibited human ErbB-2 specific CTLactivity among those against CD3, CD8, and mouse MHC class I molecule(FIG. 4). anti-CD4, anti-Dd, anti-Ld or anti-mouse MHC class II moleculeantibodies did not exhibit such activity. These results suggest that theobserved human ErbB-2 specific CTL activity was elicited by MHC classI-restricted CD8 positive T lymphocytes.

INDUSTRIAL APPLICABILITY

The vaccinal preparation of the present invention has excellentmodifying effects on immune response to antigens, in particular,activation or induction of cytotoxic T cells, and can be used as avaccine for preventive and/or therapeutic treatment of cancers, viraldiseases, and autoimmune diseases.

2 1 24 DNA synthetic construct 1 agctgcagtg atcaccatgg agct 24 2 29 DNAsynthetic construct 2 tgaattctat gtgagacttc gaagctgca 29

What is claimed is:
 1. A parenteral vaccinal preparation comprisinghydrophobized polysaccharide and antigen, wherein the hydrophobizedpolysaccharide comprises from about 0.9 to about 5 hydrophobic groupsper 100 monosaccharide units and wherein the antigen is selected fromtumor-associated antigens, viral antigens and autoantigen-reactiveT-cell receptors.
 2. The vaccinal preparation of claim 1, wherein thehydrophobic groups are selected from alkyl groups and sterol residues.3. The vaccinal preparation of claim 2, wherein the sterol residuescomprise a cholesterol residue.
 4. The vaccinal preparation of claim 1,wherein the hydrophobized polysaccharide comprises saccharide unitswhose primary hydroxyl group is represented by the following formula:—O—(CH₂)_(m)CONH(CH₂)_(n)NH—CO—O—R wherein R represents an alkyl groupor a sterol residue; m represents 0 or 1; and n represents a positiveinteger.
 5. The vaccinal preparation of claim 1, wherein the antigen isselected from antigenic proteins having a molecular weight of about2,000 to about 100,000.
 6. The vaccinal preparation of claim 5, whereinthe antigen is capable of at least one of activation and induction ofcytotoxic T-cells.
 7. The vaccinal preparation of claim 1, wherein thepreparation is free of phospholipids.
 8. The vaccinal preparation ofclaim 1, further comprising at least one of an excipient, a carrier anda diluent suitable for parenteral administration.
 9. A parenteralvaccinal preparation comprising hydrophobized polysaccharide andantigen, wherein the hydrophobized polysaccharide comprises hydrophobicgroups in a ratio of modifications to molecule of about 5% by weight orless and wherein the antigen is selected from tumor-associated antigens,viral antigens and autoantigen-reactive T-cell receptors.
 10. Thevaccinal preparation of claim 9, wherein the antigen is encapsulated bymicroparticles of aggregated hydrophobized polysaccharide.
 11. Thevaccinal preparation of claim 9, wherein the polysaccharide is selectedfrom pullulan and mannan.
 12. A microparticle for use in a vaccinalpreparation, the microparticle consisting essentially of hydrophobizedpolysaccharide and antigen selected from tumor-associated antigens,viral antigens and autoantigen-reactive T-cell receptors.
 13. Themicroparticle of claim 12, wherein the hydrophobized polysaccharide isaggregated.
 14. The microparticle of claim 13, wherein the aggregatedhydrophobized polysaccharide encapsulates said antigen.
 15. Themicroparticle of claim 13, wherein the hydrophobized polysaccharidecomprises from about 0.9 to about 5 hydrophobic groups per 100monosaccharide units.
 16. A parenteral vaccinal preparation comprisingan antigen and an adjuvant, wherein the adjuvant comprises ahydrophobized polysaccharide having from about 0.9 to about 5hydrophobic groups per 100 monosaccharide units and wherein the antigenis selected from tumor-associated antigens, viral antigens andautoantigen-reactive T-cell receptors.
 17. The vaccinal preparation ofclaim 16, wherein the polysaccharide is selected from mannan andpullulan.
 18. A method of immunization which comprises parenterallyadministering to a mammal a vaccinal preparation comprisinghydrophobized polysaccharide and antigen.
 19. The method of claim 18,wherein the antigen is capable of at least one of induction andactivation of cytotoxic T-cells.
 20. The method of claim 19, wherein thecytotoxic T-cell is antigen-specific.
 21. The method of claim 18,wherein the hydrophobized polysaccharide comprises from about 0.9 toabout 5 hydrophobic groups per 100 monosaccharide units.
 22. The methodof claim 18, wherein the hydrophobic groups are selected from alkylgroups and sterol residues.
 23. The method of claim 22, wherein thesterol residues comprise a cholesterol residue.
 24. The method of claim18, wherein the antigen is selected from antigenic proteins having amolecular weight of from about 2,000 to about 100,000.
 25. The method ofclaim 18, wherein the antigen comprises erbB-2 protein.
 26. The methodof claim 18, wherein the amount of antigen per administration is fromabout 50 to about 150 μg.
 27. The method of claim 26, comprising fromabout one to about four administrations.
 28. The method of claim 27,wherein the administration is subcutaneous.
 29. A method of preventing acondition selected from a viral disease and an autoimmune disease, in amammal, wherein the method comprises parenterally administering to saidmammal a pharmaceutical preparation comprising hydrophobizedpolysaccharide and antigen.
 30. A method of treating a conditionselected from cancer, a viral disease and an autoimmune disease, in amammal in need of such treatment, wherein the method comprisesparenterally administering to said mammal an effective amount of avaccinal preparation comprising hydrophobized polysaccharide andantigen.
 31. The method of claim 30 wherein the condition is cancer. 32.A process for making a vaccinal preparation comprising a hydrophobizedpolysaccharide and an antigen, the process comprising mixingmicroparticles of aggregated hydrophobized polysaccharide and antigenand subjecting the resultant mixture to gel chromatography.
 33. Theprocess of claim 32, wherein said microparticles are made by a processcomprising sonicating a solution of hydrophobized polysaccharide andfiltering the sonicated solution through at least one membrane.
 34. Avaccinal preparation comprising hydrophobized polysaccharide andantigen, wherein the hydrophobized polysaccharide comprises from about0.9 to about 5 hydrophobic groups per 100 monosaccharide units andwherein the antigen is selected from tumor-associated antigens.
 35. Thevaccinal preparation of claim 34, wherein the hydrophobic groups areselected from alkyl groups and sterol residues.
 36. The vaccinalpreparation of claim 35, wherein the sterol residues comprise acholesterol residue.
 37. The vaccinal preparation of claim 34, whereinthe hydrophobized polysaccharide comprises saccharide units whoseprimary hydroxyl group is represented by the following formula:—O—(CH₂)_(m)CONH(CH₂)_(n)NH—CO—O—R wherein R represents an alkyl groupor a sterol residue; m represents 0 or 1; and n represents a positiveinteger.
 38. The vaccinal preparation of claim 37, wherein thepolysaccharide is selected from pullulan and mannan.
 39. A process formaking a pharmaceutical composition comprising at least one activeingredient, comprising combining the at least one active ingredient withan adjuvant, wherein the adjuvant comprises a hydrophobizedpolysaccharide having from about 0.9 to about 5 hydrophobic groups per100 monosaccharide units.
 40. The process of claim 39, wherein thehydrophobic groups are selected from alkyl groups and sterol residues.41. The process of claim 39, wherein the hydrophobized polysaccharidecomprises saccharide units whose primary hydroxyl group is representedby the following formula: —O—(CH₂)_(m)CONH(CH₂)_(n)NH—CO—O—R wherein Rrepresents an alkyl group or a sterol residue; m represents 0 or 1; andn represents a positive integer.
 42. A vaccinal preparation comprisinghydrophobized polysaccharide and antigen, wherein the hydrophobizedpolysaccharide is selected from (a) mannan having a molecular weight ofabout 55,000 and being modified with about 2.3 cholesterol residues per100 monosaccharide units and (b) pullulan having a molecular weight ofabout 108,000 and being modified with about 0.9 cholesterol residues per100 monosaccharide units and wherein the antigen is erbB-2 protein. 43.The vaccinal preparation of claim 42, wherein the vaccinal preparationis a parenteral vaccinal preparation.
 44. The vaccinal preparation ofclaim 43, further comprising at least one of an excipient, a carrier anda diluent suitable for parenteral administration.
 45. The vaccinalpreparation of claim 42, wherein the preparation is free ofphospholipids.
 46. A complex comprising erbB-2 protein and ahydrophobized polysaccharide selected from (a) mannan having a molecularweight of about 55,000 and being modified with about 2.3 cholesterolresidues per 100 monosaccharide units and (b) pullulan having amolecular weight of about 108,000 and being modified with about 0.9cholesterol residues per 100 monosaccharide units.
 47. A microparticlefor use in a vaccinal preparation, the microparticle comprising erbB-2protein and a hydrophobized polysaccharide selected from (a) mannanhaving a molecular weight of about 55,000 and being modified with about2.3 cholesterol residues per 100 monosaccharide units and (b) pullulanhaving a molecular weight of about 108,000 and being modified with about0.9 cholesterol residues per 100 monosaccharide units.
 48. A method ofimmunization which comprises parenterally administering to a mammal avaccinal preparation comprising erbB-2 protein and a hydrophobizedpolysaccharide selected from (a) mannan having a molecular weight ofabout 55,000 and being modified with about 2.3 cholesterol residues per100 monosaccharide units and (b) pullulan having a molecular weight ofabout 108,000 and being modified with about 0.9 cholesterol residues per100 monosaccharide units.
 49. The method of claim 48, wherein the amountof erbB-2 protein per administration is from about 50 to about 150 μg.50. The method of claim 49, comprising from about one to about fouradministrations.
 51. The method of claim 48, wherein the administrationis subcutaneous.
 52. A method of inducing cytotoxic T-cells whichcomprises administering to a mammal a vaccinal preparation comprisingerbB-2 protein and a hydrophobized polysaccharide selected from (a)mannan having a molecular weight of about 55,000 and being modified withabout 2.3 cholesterol residues per 100 monosaccharide units and (b)pullulan having a molecular weight of about 108,000 and being modifiedwith about 0.9 cholesterol residues per 100 monosaccharide units.
 53. Amethod of activating cytotoxic T-cells which comprises administering toa mammal a vaccinal preparation comprising erbB-2 protein and ahydrophobized polysaccharide selected from (a) mannan having a molecularweight of about 55,000 and being modified with about 2.3 cholesterolresidues per 100 monosaccharide units and (b) pullulan having amolecular weight of about 108,000 and being modified with about 0.9cholesterol residues per 100 monosaccharide units.
 54. A method oftreating cancer in a mammal in need of such treatment, wherein themethod comprises parenterally administering to said mammal atherapeutically effective amount of a vaccinal preparation comprisingerbB-2 protein and a hydrophobized polysaccharide selected from (a)mannan having a molecular weight of about 55,000 and being modified withabout 2.3 cholesterol residues per 100 monosaccharide units and (b)pullulan having a molecular weight of about 108,000 and being modifiedwith about 0.9 cholesterol residues per 100 monosaccharide units.
 55. Aprocess for making a vaccinal preparation, the process comprising mixingmicroparticles of erbB-2 protein and aggregated hydrophobizedpolysaccharide selected from (a) mannan having a molecular weight ofabout 55,000 and being modified with about 2.3 cholesterol residues per100 monosaccharide units and (b) pullulan having a molecular weight ofabout 108,000 and being modified with about 0.9 cholesterol residues per100 monosaccharide units, and subjecting the resultant mixture to gelchromatography.
 56. The process of claim 55, wherein said microparticlesare made by a process comprising sonicating a solution of hydrophobizedpolysaccharide and filtering the sonicated solution through at least onemembrane.